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. 2009 Jul;175(1):74–83. doi: 10.2353/ajpath.2009.080884

Figure 5.

Figure 5

In vitro ubiquitination assay of TRADD in UUO kidney extracts (A) and in vivo TRADD-ubiquitination in HEK 292 cells (B). A: Renal extracts collected from sham-operated and UUO kidneys at day 7 were incubated with immunopurified FLAG-tagged TRADD (FLAG-TRADD) protein for 60 minutes at 37°C. The resulting immunoprecipitates were washed thoroughly four times with cold lysis buffer, then FLAG-TRADD was eluted by FLAG peptide, and the assay products were subjected to immunoblot analysis using anti-ubiquitin antibody. Significant ladders were observed when FLAG-TRADD protein was incubated with renal extracts collected from UUO kidneys, but only a few weak ladders were noted when FLAG-TRADD protein was incubated with the extracts from sham-operated kidneys. Scant ladders were observed when FLAG-TRADD protein was incubated without renal extracts. Furthermore, no significant bands were observed when anti-FLAG immunoprecipitated protein, which was derived from HEK293 cells transfected with pcDNA3 lacking FLAG-TRADD as a negative control, was incubated with renal extracts collected from UUO rat at day 7. IgG bands are indicated by an asterisk. B: pcDNA3-FLAG-TRADD plasmid was transfected together with or without pCGN-HA-tagged ubiquitin (HA-Ub) plasmid into HEK293 cells. Forty-five hours after transfection, the cells were incubated with or without 10 μmol/L MG132 for 3 hours. The immunoprecipitates obtained from the cell lysates using anti-FLAG M2 beads were washed four times with lysis buffer and subjected to double immunoprecipitation. The first complexes immunoprecipitated using anti-FLAG M2 beads were denatured by treatment with SDS sample buffer at 100°C for 7 minutes. Then, ubiquitinated FLAG-TRADD was reimmunoprecipitated with anti-FLAG M2 affinity beads, separated by SDS-PAGE (9%), and detected by immunoblot with anti-HA antibody or anti-FLAG M2 antibody. Significant ladders of polyubiquitinated TRADD were observed when FLAG-TRADD was transfected with HA-Ub, but no ladders were noted without HA-Ub transfection.